Triple negative (ER-, PR-, HER2-) breast cancers (TNBCs) lack targeted treatments, resulting in a high mortality rate. Recurrence of chemotherapy-resistant tumors as well as formation of distant metastases are both contributors to mortality within the first 3-5 years. We and others have shown that TNBCs are enriched with cancer stem-cell (CSC)- and mesenchymal features, and that the induction of an epithelial-mesenchymal- transition (EMT) promotes the de novo generation/expansion of drug-resistant CSCs. Indeed, the gene expression signature generated by induction of EMT is enriched in claudin-low breast cancers, a particular intrinsic subtype comprising nearly half of all TNBCs. We recently reported that expression of the forkhead-box transcription factor FOXC2 is uniquely induced following EMT and in CSCs, and that FOXC2 is sufficient for EMT, tumor initiation, and metastasis of TNBC cell lines and chemotherapy-resistance. Remarkably, shRNA-mediated knockdown of FOXC2 is sufficient to block induction of EMT, metastasis as well as the CSC-associated properties of tumor initiation and chemotherapy-resistance. Given these results, we reasoned that a drug discovery screen for small molecules that selectively target FOXC2-expressing CSCs will yield important chemical agents that will dramatically improve treatment of CSC-enriched TNBCs. We have performed a high-throughput small molecule screen of approximately 100,000 agents, and have identified seven candidate molecules with preferential activity against FOXC2-expressing CSCs. In this proposal, we intend to fully characterize and validate the in vitro activity of these molecules across a wide spectrum of breast cancer cell lines for their selectivity for CSCs, their ability to block induction and reverse EMT-associated tumor cell properties (including migration and invasion), and their capacity to alter CSC traits (including mammosphere formation, marker expression and colony formation). In addition, we will elucidate the relevant protein targets for these agents, by use of photo-affinity reagents followed by mass spectrometry, and also determine the functional role of these target proteins for EMT/CSC properties. Finally, we will optimize candidate molecules for in vivo delivery and acceptable pharmacokinetics, and conduct extensive preclinical testing in mice using multiple TNBC patient-derived xenografts. The results of this study will directly permit the identification of novel drugs for treating recurrent and metastatic TNBCs, and will also shed light on the molecular networks regulated by FOXC2 during its reprogramming of tumor cells towards the CSC/metastatic state. The strong collaboration between Dr. Mani and Dr. Lairson, evidenced by our extensive preliminary data, indicates a high likelihood of success. Furthermore, the extended scope of this proposal, from drug discovery using high-throughput screens, to testing in patient-derived TNBC xenografts, indicates our commitment to rapidly moving novel cancer therapeutics into clinical trials.